Organization of monoterpene biosynthesis in Mentha. Immunocytochemical localizations of geranyl diphosphate synthase, limonene-6-hydroxylase, isopiperitenol dehydrogenase, and pulegone reductase

We present immunocytochemical localizations of four enzymes involved in p-menthane monoterpene biosynthesis in mint: the large and small subunits of peppermint (Mentha x piperita) geranyl diphosphate synthase, spearmint (Mentha spicata) (-)-(4S)-limonene-6-hydroxylase, peppermint (-)-trans-isopiperitenol dehydrogenase, and peppermint (+)-pulegone reductase. All were localized to the secretory cells of peltate glandular trichomes with abundant labeling corresponding to the secretory phase of gland development. Immunogold labeling of geranyl diphosphate synthase occurred within secretory cell leucoplasts, (-)-4S-limonene-6-hydroxylase labeling was associated with gland cell endoplasmic reticulum, (-)-trans-isopiperitenol dehydrogenase labeling was restricted to secretory cell mitochondria, while (+)-pulegone reductase labeling occurred only in secretory cell cytoplasm. We discuss this pathway compartmentalization in relation to possible mechanisms for the intracellular movement of monoterpene metabolites, and for monoterpene secretion into the extracellular essential oil storage cavity.     

Development of peltate glandular trichomes of peppermint

Cryofixation and conventional chemical fixation methods were employed to examine the ultrastructure of developing peltate glandular trichomes of peppermint (Mentha x piperita). Our results are discussed in relation to monoterpene production and the mechanism of essential oil secretion. Peltate glands arise as epidermal protuberances (initials) that divide asymmetrically to produce a vacuolate basal cell, a stalk cell, and a cytoplasmically dense apical cell. Further divisions of the apical cell produce a peltate trichome with one basal cell, one stalk cell, and eight glandular (secretory) disc cells. Presecretory gland cells resemble meristematic cells because they contain proplastids, small vacuoles, and large nuclei. The secretory phase coincides with the separation and filling of the sub-cuticular oil storage space, the maturation of glandular disc cell leucoplasts in which monoterpene biosynthesis is known to be initiated, and the formation of extensive smooth endoplasmic reticulum at which hydroxylation steps of the monoterpene biosynthetic pathway occur. The smooth endoplasmic reticulum of the secretory cells appears to form associations with both the leucoplasts and the plasma membrane bordering the sub-cuticular oil storage cavity, often contains densely staining material, and may be involved with the transport of the monoterpene-rich secretion product. Associated changes in the ultrastructure of the secretory stage stalk cell are also described, as is the ultrastructure of the fragile post-secretory gland for which cryofixation methods are particularly well suited for the preservation of organizational integrity.     

Monoterpene hydrocarbon biosynthesis by isolated leucoplasts of Citrofortunella mitis

A plastid vesicle preparation isolated from exocarpium of young Citrofortunella mitis (calamondin) fruits was able to synthesise monoterpene hydrocarbons when incubated with isopentenyl pyrophosphate. The electron-microscope comparison between this organelle fraction and the various plastid classes present in the peel tissues has shown the structural identity between these plastid vesicles and the leucoplasts of the epithelial cells lining the secretory pockets. The monoterpene biosynthesis required the presence of dimethylallyl pyrophosphate, Mn²⁺ or Mg²⁺ and was increased by addition of 2-mercaptoethanol. Evidence is provided that the leucoplast vesicles act as a complete system in which occur all the successive steps involved in monoterpene hydrocarbon elaboration from isopentenyl pyrophosphate.     

Cosuppression of limonene-3-hydroxylase in peppermint promotes accumulation of limonene in the essential oil

cDNA clones encoding limonene synthase and limonene-3-hydroxylase, both driven by the CaMV 35S promoter, were independently transformed into peppermint (Menthaxpiperita) to alter the production and disposition of (-)-limonene, the first committed intermediate of essential oil biosynthesis in this species. Although both genes were constitutively expressed in leaves of transformed plants, the corresponding enzyme activities were not significantly increased in the glandular trichome sites of essential oil biosynthesis; thus, there was no effect on oil yield or composition in the regenerated plants. Cosuppression of the hydroxylase gene, however, resulted in the accumulation of limonene (up to 80% of the essential oil compared to about 2% of the oil in wild type plants), without influence on oil yield. These results indicate that limonene does not impose negative feedback on the synthase, or apparently influence other enzymes of monoterpene biosynthesis in peppermint, and suggests that pathway engineering can be employed to significantly alter essential oil composition without adverse metabolic consequences.     

Isoprenoid biosynthesis. Metabolite profiling of peppermint oil gland secretory cells and application to herbicide target analysis

Two independent pathways operate in plants for the synthesis of isopentenyl diphosphate and dimethylallyl diphosphate, the central intermediates in the biosynthesis of all isoprenoids. The mevalonate pathway is present in the cytosol, whereas the recently discovered mevalonate-independent pathway is localized to plastids. We have used isolated peppermint (Mentha piperita) oil gland secretory cells as an experimental model system to study the effects of the herbicides fosmidomycin, phosphonothrixin, methyl viologen, benzyl viologen, clomazone, 2-(dimethylamino)ethyl diphosphate, alendronate, and pamidronate on the pools of metabolites related to monoterpene biosynthesis via the mevalonate-independent pathway. A newly developed isolation protocol for polar metabolites together with an improved separation and detection method based on liquid chromatography-mass spectrometry have allowed assessment of the enzyme targets for a number of these herbicides.     

ULTRASTRUCTURAL DEVELOPMENT OF CAPITATE GLANDULAR HAIRS OF CANNABIS SATIVA L. (CANNABACEAE)

The capitate-sessile and capitate-stalked glands of the glandular secretory system in Cannabis, which are interpreted as lipophilic type glandular hairs, were studied from floral bracts of pistillate plants. These glands develop a flattened multicellular disc of secretory cells, which with the extruded secretory product forms the gland head and the auxiliary cells which support the gland head. The secretory product accumulates beneath a sheath derived from separation of the outer wall surface of the cellular disc. The ultrastructure of secretory cells in pre-secretory stages is characterized by a dense ground plasm, transitory lipid bodies and fibrillar material, and well developed endoplasmic reticulum. Dictyosomes and dictyosome-derived secretory vesicles are present, but never abundant. Secretory stages of gland development are characterized by abundant mitochondria and leucoplasts and by a large vacuolar system. Production of the secretory product is associated with plastids which increase in number and structural complexity. The plastids develop a paracrystalline body which nearly fills the mature plastid. Material interpreted as a secretion appears at the surface of plastids, migrates, and accumulates along the cell surface adjoining the secretory cavity. Extrusion of the material into the secretory cavity occurs directly through the plasma membrane-cell wall barrier.     

Leucoplasts: a distinct kind of organelles lacking typical 70S ribosomes and free thylakoids

Non-pigmented plastids were observed in fully differentiated cells from leaves and stem tissues of various species. Although showing important differences in size and shape, these plastids exhibit permanent structural features which allow to get them together as a distinct kind of organelles: the leucoplasts. Leucoplasts are distinct from the proplastids and every intermediate stage of plastid differentiation, from white chromoplasts and tuber amyloplasts. Mature leucoplasts do not contain an autonomous central system of thylakoids structurally independent from the envelope and, therefore, are never green. However, the envelope inner membrane invaginates within the plastid a cisternal or tubular stroma reticulum connected with the intermembrane space of the envelope. In addition, the leucoplast stroma is often less dense than chloroplasts stroma and contain several nucleoids with DNA fibrils. However, 70S ribosomes either scattered in the stroma or attached to the stroma reticulum or the envelope are not visible in ultrathin sections of leucoplasts stained with uranyl and lead. The existence of more discrete particles as dense as ribosomes is suggested. The relationship between the absence of ribosomes and thylakoids is discussed. Except for their specific role in C10 monoterpene synthesis in glandular cells, the functions of leucoplasts in plant cells remains largely up to now a matter of conjecture.     

Characterization of a novel laccase produced by the wood-rotting fungus Phellinus ribis

(+)-Menthofuran is an undesirable monoterpenoid component of peppermint (Mentha x piperita) essential oil that is derived from the alpha,beta-unsaturated ketone (+)-pulegone. Microsomal preparations, from the oil gland secretory cells of a high (+)-menthofuran-producing chemotype of Mentha pulegium, transform (+)-pulegone to (+)-menthofuran in the presence of NADPH and molecular oxygen, implying that menthofuran is synthesized by a mechanism analogous to that of mammalian liver cytochrome P450s involving the hydroxylation of the syn-methyl group of (+)-pulegone, spontaneous intramolecular cyclization to the hemiketal, and dehydration to the furan. An abundant cytochrome P450 clone from a peppermint oil gland cell cDNA library was functionally expressed in Saccharomyces cerevisiae and Escherichia coli and shown to encode the (+)-menthofuran synthase (i.e., (+)-pulegone-9-hydroxylase). The full-length cDNA contains 1479 nucleotides, and encodes a protein of 493 amino acid residues of molecular weight 55,360, which bears all of the anticipated primary structural elements of a cytochrome P450 and most closely resembles (35% identity) a cytochrome P450 monoterpene hydroxylase, (+)-limonene-3-hydroxylase, from the same source. The availability of this gene permits transgenic manipulation of peppermint to improve the quality of the derived essential oil.     

A cDNA clone for 3-carene synthase from Salvia stenophylla

The essential oil of Salvia stenophylla contains (+)-3-carene as the principal monoterpene component. Using an enriched cDNA library prepared from mRNA isolated from S. stenophylla peltate glandular trichomes, and a homology-based cloning strategy, a full-length cDNA was isolated that encoded a preprotein of 69.7 kDa which resembled a monoterpene synthase in sequence. Heterologous expression of the gene in Escherichia coli provided a soluble recombinant enzyme capable of catalyzing the divalent metal ion-dependent conversion of geranyl diphosphate to (+)-3-carene and to lesser amounts of limonene, myrcene, 4-carene and beta-phellandrene. This multiple-product synthase is responsible for the production of all of the essential oil monoterpenes of S. stenophylla.     

Molecular cloning of geranyl diphosphate synthase and compartmentation of monoterpene synthesis in plant cells

The nature of isoprenoids synthesized in plants is primarily determined by the specificity of prenyltransferases. Several of these enzymes have been characterized at the molecular level. The compartmentation and molecular regulation of geranyl diphosphate (GPP), the carbon skeleton that is the backbone of myriad monoterpene constituents involved in plant defence, allelopathic interactions and pollination, is poorly understood. We describe here the cloning and functional expression of a GPP synthase (GPPS) from Arabidopsis thaliana. Immunohistological analyses of diverse non-secretory and secretory plant tissues reveal that GPPS and its congeners, monoterpene synthase, deoxy-xylulose phosphate synthase and geranylgeranyl diphosphate synthase, are equally compartmentalized and distributed in non-green plastids as well in chloroplasts of photosynthetic cells. This argues that monoterpene synthesis is not solely restricted to specialized secretory structures but can also occur in photosynthetic parenchyma. These data provide new information as to how monoterpene biosynthesis is compartmentalized and induced de novo in response to biotic and abiotic stress in diverse plants.     

Ultrastructural studies on the secretory cavities ofCitrus deliciosa ten. II. Development of the essential oil-accumulating central space of the gland and process of active secretion

Summary Oil glands ofCitrus deliciosa are multicellular secretory structures, globular to oval in shape, in the centre of which an essential oil-accumulating space is formed. Opening of this space begins from a single cell. It undergoes lysis which later extends to the neighbouring gland cells.Secretory material in form of droplets is produced in plastids, from where it is transported to the parietal cytoplasm of the secretory cells via numerous ER-elements. After fusion of the ER-membranes with the plasmalemma, the exudate reaches the apoplast, through which it is driven to the central cavity of the gland.Peripheral cells of the secretory complex are modified into a protective sheath with thick walls and large vacuoles, while their plastids are differentiated from leucoplasts into typical amyloplasts.     

Biosynthesis of the Monoterpenes Limonene and Carvone in the Fruit of Caraway : I. Demonstration of Enzyme Activities and Their Changes with Development

The biosynthesis of the monoterpenes limonene and carvone in the fruit of caraway (Carum carvi L.) proceeds from geranyl diphosphate via a three-step pathway. First, geranyl diphosphate is cyclized to (+)-limonene by a monoterpene synthase. Second, this intermediate is stored in the essential oil ducts without further metabolism or is converted by limonene-6-hydroxylase to (+)-trans-carveol. Third, (+)-trans-carveol is oxidized by a dehydrogenase to (+)-carvone. To investigate the regulation of monoterpene formation in caraway, we measured the time course of limonene and carvone accumulation during fruit development and compared it with monoterpene biosynthesis from [U-¹⁴C]Suc and the changes in the activities of the three enzymes. The activities of the enzymes explain the profiles of monoterpene accumulation quite well, with limonene-6-hydroxylase playing a pivotal role in controlling the nature of the end product. In the youngest stages, when limonene-6-hydroxylase is undetectable, only limonene was accumulating in appreciable levels. The appearance of limonene-6-hydroxylase correlates closely with the onset of carvone accumulation. At later stages of fruit development, the activities of all three enzymes declined to low levels. Although this correlates closely with a decrease in monoterpene accumulation, the latter may also be the result of competition with other pathways for substrate.     

Limonene production in tobacco with Perilla limonene synthase cDNA

Limonene synthase (LS) catalyses the stereo-specific cyclization of geranyl diphosphate (GPP) to form a monocyclic monoterpene, limonene. In an attempt to engineer monoterpene biosynthesis, three expression constructs of LS cDNA of Perilla frutescens, which were designed to be localized in either the plastid, the cytosol or the endoplasmic reticulum (ER), were introduced into tobacco in order to examine differences in enzyme activity and the productivity of limonene. High and moderate enzyme activity, respectively, was observed for plastid- and cytosol-localized LS, whereas no enzyme activity was seen for ER-localized LS, suggesting that the plastid is the preferred compartment for LS, while LS may also have an active form in the cytosol. The formation of limonene in vivo was confirmed by gas chromatography-mass spectrometry (GC-MS) in leaf extracts of both plastid- and cytosol-localized LS transgenic plants. The amount of limonene in plastid-localized LS transgenic plants was 143 ng g-1 fresh wt, whereas that in the cytosol-type was 40 ng g-1 fresh wt, and these limonene contents increased by 2.7-fold and 3.0-fold, respectively, with the addition of methyl jasmonate. The headspace analyses showed that the plastid- and the cytosol-localized LS transgenic plants (12 cm high) emitted 390 ng and 515 ng limonene per month, respectively. The possibility of genetically engineering monoterpene production is discussed.     

Regulation of leucoplast morphology in roots: Interorganellar signaling from mitochondria?

The appearance of leaf mesophyll chloroplasts in angiosperms is characterized by their uniform and static shape, which is molded by symmetric division of the preexisting organelles, involving three prokaryote-derived proteins: the division executor protein, FtsZ, and the division site positioning proteins, MinD and MinE. On the other hand, noncolored plastids in roots, where the involvement of the known chloroplast division factors in plastid morphogenesis is yet unclear, are morphologically heterogeneous and transform dynamically. This is further emphasized by the active formation of long tubular protrusions called stromules from the main body of those plastids. Molecular regulation and physiological significance of such dynamic morphology of root plastids also remain unknown. In this context, we have recently demonstrated that the mitochondrial respiratory inhibitor antimycin A induces rapid and reversible filamentation of root plastids (leucoplasts) in Arabidopsis thaliana. In contrast, the same treatment with antimycin A did not affect the morphology of amyloplasts in the columella cells at the root tip. The alternative oxidase inhibitor salicylhydroxamic acid suppresses the antimycin-induced plastid filamentation, perhaps implying an alternative oxidase-mediated interorganellar signaling between the mitochondria and the leucoplasts in the root cells. Our data may provide some clues as to how the formation of stromules is initiated.Key words: antimycin A, interorganellar crosstalk, plastid morphology, respiration, stress response, stromule     

In vivo analysis of the role of atTic20 in protein import into chloroplasts

The import of nucleus-encoded preproteins into plastids requires the coordinated activities of membrane protein complexes that facilitate the translocation of polypeptides across the envelope double membrane. Tic20 was identified previously as a component of the import machinery of the inner envelope membrane by covalent cross-linking studies with trapped preprotein import intermediates. To investigate the role of Tic20 in preprotein import, we altered the expression of the Arabidopsis Tic20 ortholog (atTic20) by antisense expression. Several antisense lines exhibited pronounced chloroplast defects exemplified by pale leaves, reduced accumulation of plastid proteins, and significant growth defects. The severity of the phenotypes correlated directly with the reduction in levels of atTic20 expression. In vitro import studies with plastids isolated from control and antisense plants indicated that the antisense plastids are defective specifically in protein translocation across the inner envelope membrane. These data suggest that Tic20 functions as a component of the protein-conducting channel at the inner envelope membrane.     

FINE STRUCTURE OF PROTEIN-STORING PLASTIDS IN BEAN ROOT TIPS

The fine structure of leucoplasts in root tip cells of Phaseolus vulgaris L. has been studied in material fixed in glutaraldehyde followed by osmium tetroxide and poststained in uranyl acetate and lead citrate. Plastid development has been followed from the young stages in and near the meristematic region, through an ameboid stage, to the larger forms with more abundant storage products in the outermost cells. The plastids contain a dense stroma penetrated by tubules and cisternae arising from the inner membrane of the plastid envelope. Also located in the stroma are lamellae, ribosome-like particles, phytoferritin granules, and fine fibrils in less dense regions. In some elongate plastids microfilaments run lengthwise in the stroma near the surface. The same plastids store both starch and protein, but in a strikingly different manner. The starch is deposited in the stroma, while the protein always is accumulated within membrane-bounded sacs. These sacs arise as outgrowths from a complex of interconnected tubules which in turn appears to originate by coalescence and proliferation of tubules and cisternae arising from the inner plastid membrane. This "tubular complex" bears a strong resemblance to the prolamellar body of etiolated chloroplasts, but is smaller and ordinarily less regularly organized, and is apparently light-insensitive. Crystallization of the protein commonly occurs in the sacs and occasionally takes place within the tubules of the complex as well. The fine structure of the leucoplasts is discussed in relation to that of etiolated chloroplasts. Suggestions are made concerning the function of the tubular complex, role of the ameboid plastid forms, and manner of accumulation of the storage protein in the plastids.     

Spatial organization and volume density of leucoplasts in pine secretory cells

Summary Spatial reconstructions of pine leucoplasts were obtained from thin serial sections of resin duct cells. Plastid volume and envelope surface were estimated using morphometric methods and compared to chloroplasts of adjacent parenchyma cells.The high number of plastid sections in secretory cells is not related to leucoplast division occurring during the secretory stage, but to the differentiation of complex, amoeboid plastids, closely imbricated with each other. Furthermore, there is no leucoplast network resulting from the fusion of preexisting plastids.In contrast to chloroplasts, leucoplast shape is not ruled by a single morphogenetic program but results from rapid growth in all directions, filling most of the free cytoplasmic space. The plastid surface is enclosed by a continuous sheath of endoplasmic reticulum.The leucoplast volume per cytoplasm volume unit in a secretory cell is 2.5 times that of chloroplasts in a parenchyma cell. Owing to the overlapping of plastid structures, the leucoplast surface is more than 3 times larger than that of chloroplasts with the same space factor. The active production of terpenes during the short period of secretion is supported by a very specialized structure, the leucoplastidome, where the biosynthetic process is optimized by an increase in plastid volume and enlargement of the plastid surface allowing rapid processing of precursors and free outflow of the end products.